On  tho  Chemical  Constitution 
of  the  Proteins   of  Wheat 
Flour  and  its   Relation  to 
Baking  Strength. 

Morri  s    J  .    Blish 


B    5    7T7    T73 


•»oja  pjojxbo 


KXCHANOE 


On  the  Chemical  Constitution  of  the 

Proteins  of  Wheat  Flour  and  Its 

Relation  to  Baking  Strength 


A  THESIS  SUBMITTED  TO  THE  FACULTY  OF  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 

BY 

MORRIS  J.  BLISH 

IN  PARTIAL  FULFILLMENT    OF  THE   REQUIREMENTS  FOR 
THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 

June,  1915 


Easton,  Pa.: 

EscHENBACH  Printing  Co. 

1916 


On  the  Chemical  Constitution  of  the 

Proteins  of  Wheat  Flour  and  Its 

Relation  to  Baking  Strength 


A  THESIS  SL'BMITTED  TO  THE  FACULTY  OP  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 

BY 

MORRIS  J.  BLISH 

IN  PARTIAL  FULFILLMENT    OF  THE    REQUIREMENTS  FOR 
THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 

June,  1915 


Easton,  Pa.: 

EscHENBACH  Printing  Co. 

1916 


BXOh<^.-,.j4B 


ON  THE  CHEMICAL  CONSTITUTION  OF  THE  PROTEINS 

OF  WHEAT  FLOUR  AND  ITS  RELATION  TO 

BAKING  STRENGTH 

By  M.  J.   Bush 

Received  June  7,  1915 

INTRODUCTION 

The  most  generally  accepted  definition  of  "baking 
strength"  of  a  wheat  flour  is  that  put  forward  by 
Humphries  and  Biffen/  in  1907,  which  states  that  a 
"strong  wheat  is  one  which  yields  flour  capable  of 
making  large,   well-piled  loaves;"  a  definition  similar 

to  that  of  Jago,"  who  states  that  "strength is 

defined  as  the  measure  of  the  capacity  of  the  flour  for 
producing  a  bold,  large-volumed,  well-risen  loaf." 
Since  the  value  of  wheat  (other  things  being  equal) 
depends  on  the  so-called  "strength"  of  the  flour  which 
may  be  made  from  it,  it  is  obviously  of  great  importance 
that  complete  knowledge  be  obtained  concerning  the 
factors  which  cause  strength,  and  to  this  end  an 
enormous  amount  of  scientific  work  has  been  done, 
especially  during  the  last  twenty  years.  In  spite  of 
the  fact  that  some  of  the  foremost  investigators  of 
the  world  have  bent  their  energies  to  this  task,  the 
problem  is  not  yet  completely  solved,  although  con- 
siderable light  has  been  thrown  on  the  subject.  It  is 
not  yet  possible  to  correlate  baking  strength  with  any 
chemical  or  physical  factor  to  such  an  extent  that  a 
simple  laboratory  test  or  group  of  tests  will  always 
furnish  an  infallible  guide,  but  it  is  necessary  to  mill 
the  wheat  into  flour  and  have  a  sample  of  it  actually 
baked  into  a  loaf  of  bread  by  an  expert  baker,  before 
its  strength  can  be  accurately  ascertained. 

FACTORS     WHICH     MAY     INFLUENCE     BAKING     STRENGTH 

Almost  every  known  constituent,  or  group  of  con- 
stituents, and  almost  every  known  physical  and  chemical 
property  of  flour  has  been  investigated  with  respect 
to  its  possible  relation  to  baking  strength,  but  as  yet 
no  one  is  believed  to  have  discovered  a  limiting  factor 
or  group  of  factors  which  completely  solves  the  prob- 
lem. Moreover,  there  is  a  general  disagreement 
among  many  of  the  leading  investigators  as  to  the 
importance  which  should  be  attached  to  each  factor 

1  "The   Improvement  of  English  Wheat,"   Jour.   Agr.   Set.,   2   (1907), 
1-16. 

2  "Technology  of  Bread  Making,"  Chap.  XV,  p.  291,  1911. 


457923 


4 
or  set  of  factors,  and  two  workers  frequently  have 
arrived  at  exactly  opposite  conclusions  after  having 
investigated  practically  the  same  problem;  however, 
much  of  this  confusion  is  caused  by  the  use  of  different 
methods  of  analysis. 

A  brief  review  of  some  of  the  more  important  work 
which  has  been  done  will  serve  to  bear  out  the  pre- 
ceding statement,  as  well  as  to  indicate  the  many  sides 
from  which  the  question  of  flour  strength  has  been 
studied. 

GLiADiN-GLUTENiN  RATIO — As  soon  as  Osbornc  and 
Voorhees,^  in  1893,  established  the  composition  and 
properties  of  the  wheat  proteins,  attention  was  at- 
tracted to  gliadin  and  glutenin,  the  two  conspicuous 
and  characteristic  proteins  of  wheat,  which  were 
shown  to  make  up  the  gluten,  the  more  or  less  elastic 
binding  material  which  enables  flour  to  be  made  into 
the  dough  with  its  characteristic  elastic,  gas-retaining 
property,  and  which  may  be  separated  from  the  starch 
and  soluble  proteins  by  the  well-known  process  of 
washing  the  dough  in  a  stream  of  water.  Fleurent,' 
in  1896,  claimed  that  flour  strength  depends  on  the 
proportion  of  gliadin  to  glutenin  present  in  the  gluten 
of  the  flour.  He  concluded  from  his  experiments 
that  the  optimum  ratio  was  75  parts  of  gliadin  to  25 
of  glutenin  or  3:1.  He  assigned  certain  limits,  outside 
of  which  flours  were  said  to  be  of  poor  baking  quality. 
Snyder,^  in  1899,  published  similar  results,  although 
he  fixed  his  ideal  ratio  at  65:35.  He  also  states* 
that  the  quality  rather  than  the  quantity  of  gluten  is 
the  important  factor,  because  he  was  able  to  add  up 
to  20  per  cent  starch  to  flour  without  decreasing  its 
baking  quality.  Regarding  the  quantity  of  gluten  in 
flour,  the  amount  of  gliadin  present,  the  ratio  of  gliadin 
to  glutenin,  and  the  relation  of  these  to  baking  quality, 
it  suffices  to  say  that  the  results  of  different  investi- 
gators quite  frequently  are  not  concordant.  However, 
as  mentioned  before,  this  is  in  a  considerable  measure 
due  to  different  analytical  methods  employed  by  differ- 
ent workers. 

CRUDE  GLUTEN — The  crudc  gluten  determination, 
which  consists  essentially  of  washing  the  gluten  free 
from  starch  and  soluble  material  by  means  of  water, 
and  weighing  the  gluten,  both  in  a  wet  and  dry  state, 

»  "The  Proteids  of  the  Wheat  Kernel,"  Am.  Chetn.  J..  16  (1893). 
392-471;  Ibid.,  16  (1894).  524-535. 

'  "Sur  une  method  chimique  d'appreciation  de  la  valeur  boulangere 
des  farines  de  bl6."  Compt.  rend..  123  (1896),  755-758. 

»  Minn.  Exp.  Sta.  Bull.,  62  (1899). 

«  U.  S.  Dept.  Agr.,  Bull.  101  (1901). 


5 

was  for  a  long  time  considered  of  great  value,  but 
Snyder  and  Norton/  in  1906,  Chamberlain/  in  1906, 
and  others  showed  that  it  gave  but  little  information 
which  might  not  be  gained  from  a  determination  of 
total  nitrogen  or  alcohol-soluble  nitrogen.  Neverthe- 
less, it  is  still  used  extensively  by  millers  and  bakers, 
and  in  technical  laboratories. 

PHYSICAL    STATE    OF    GLUTEN    AND    SUGAR    CONTENT 

In  1907,  Wood'  published  the  results  of  a  thorough 
and  systematic  study  of  the  chemistry  of  fiour  strength. 
He  concluded  that  there  is  no  difference  in  the  chemical 
constitution  of  gliadin  and  glutenin  from  strong  and 
weak  flours,  and  decided  that  strength  (particularly 
shape  of  loaf)  is  much  more  closely  related  to  the 
physical  state  of  gluten,  which  in  turn  is  profoundly 
affected  by  the  presence  of  electrolytes.  He  showed 
that  minute  quantities  of  acids  and  bases  tend  to 
"disperse"  gluten,  making  it  weak'  and  inelastic, 
while  small  quantities  of  neutral  salts  have  the  opposite 
and  consequently  beneficial  effect.  Furthermore,  he 
found  that  the  volume  of  a  loaf  of  bread  is  proportional 
to  the  rate  of  carbon  dioxide  evolution  resulting  from 
diastatic  activity  of  yeast  in  the  later  stages  of  fer- 
mentation. In  other  words,  he  concludes  that  loaf 
volume  depends  on  the  amount  of  available  sugar  in 
the  later  stages  of  fermentation.  Alway  and  Hartzell,* 
in  1909,  however,  performed  experiments  which  led 
them  to  say,  in  contrast  to  Wood's  findings,  "there  is 
clearly  no  direct  connection  shown  between  the  size 
of  the  loaf  and  the  volume  of  gas  evolved.  The  thir- 
teen flours  which  gave  the  largest  loaves  evolved  on 
the  average  somewhat  less  gas  than  the  other  thirteen 
flours."  Shutt^  states  that  from  his  experimental 
evidence  he  was  unable  to  find  any  relation  between 
size  of  loaf  and  sugar  content. 

ENZYMES — Comparatively  less  study  has  been  made 
of  the  enzymes  of  flour  and  their  relation  to  strength. 
Perhaps  the  most  prominent  work  in  this  field  is  that 
which  was  done  simultaneously  but  independently  by 
Baker   and    Hulton,^   and   by   Ford   and    Guthrie,^   in 

»  "Crude  Gluten."  J.  Am   Chem.  Soc,  28  (1906),  8-25. 

2  "Properties  of  Wheat  Proteins,"  Ibid.,  28  (1906),  1657-1667. 

'  "The  Chemistry  of  Strength  of  Wheat  Flour,"  Jour.  Agr.  Set.,  2 
(1907),   139-161   and  267-277. 

*  Neb.  Exp.  Sta.,  23rd  Annual  Report,  1909. 

5  "Flour- — the  Relationship  of  Composition  to  Bread  Making  Value," 
Canadian  Miller  and  Cerealist,  5  (1913),  176-178. 

'  "Conditions  Affecting  the  Strength  of  Wheaten  Flour,"  Jour.  Soc. 
Chem.  Ind.,  27  (1908),  368-376. 

'  "The  Amylolytic  and  Proteolytic  Ferments  of  Wheaten  Flour  and 
Relation  to  Baking  Value,"  Jour.  Soc.  Chem.  Ind.,  27  (1908),  389-393. 


iQoS.  They  point  out  that  both  proteoclastic  and 
amyloclastic  enzymes  are  present  in  flour  and  in  many 
instances  may  exert  a  profound  influence  on  its  bread- 
making  qualities.  Baker  and  Hulton  state  that 
"it  is  obvious  that  the  strength  of  a  flour  must  be 
closely  connected  with  the  gluten,  although  no  doubt 
the  presence  of  enzymes,  soluble  carbohydrates,  and 
mineral  constituents  all  play  a  part."  Koch,*  in 
1914,  found  no  difference  in  the  quantity  of  diastase 
in  strong  and  weak  flours,  after  extracting  them  with 
water  at  0°  according  to  the  method  of  Thatcher 
and  Koch. 2 

CONCENTRATION      OF      HYDROGEN      IONS H.      JeSSen- 

Hansen,^  in  1911,  finds  a  close  relationship  between 
the  concentration  in  hydrogen  ions  and  baking  strength, 
and  asserts  that  there  is  an  optimum  hydrogen-ion 
concentration  for  flour,  the  poorer  flours  having  lower 
concentrations.  He  attributes  the  beneflcial  effects 
of  neutral  salts  and  "flour  improvers"  on  flour  to  the 
fact  that  they  raise  the  hydrogen  ion  concentra- 
tion. 

SOLUBLE  PROTEINS — There  does  not  seem  to  have 
been  a  very  considerable  amount  of  work  done  regard- 
ing the  r61e  of  the  soluble  proteins  as  a  factor  in  baking 
strength.  Snyder,^  in  1897,  says  "When  any  of  the 
wheat  proteids  except  gliadin  or  glutenin  are  extracted 
the  expanding  and  bread-making  qualities  of  the  flour 
are  not  affected."  The  conclusions  of  Bremer,^ 
in  1907,  are  also  to  the  effect  that  the  soluble  proteins 
have  little  bearing  on  flour  strength.  Rousseaux 
and  Sirot,^  in  1913,  consider  the  ratio  of  total  nitrogen 
to  soluble  nitrogen  as  a  valuable  index  to  baking  value 
and  have  determined  an  ideal  ratio  for  flours  according 
to  their  method,  as  well  as  the  limits  between  which 
strong  flours  must  fall  in  this  respect. 

GENERAL  CONSIDERATIONS — Numcrous  Other  results 
of  careful  and  valuable  research  might  be  cited,  but 
the  above  serve  to  indicate  the  confusion  existing  in 

'  "The  Diastase  and  Invertase  Content  of  Wheat  Flour  and  Their 
Relation  to  Baking  Strength."  Thesis  for  Master's  Degree,  University  of 
Minnesota.  June,   1914. 

'  "The  Quantitative  Extraction  of  Diastases  from  Plant  Tissues," 
J.  Am.  Chem.  Soc,  36  (1914),  759-770. 

'  "Studies  on  Wheat  Flour.  Influence  of  H-ion  Concentration  on 
Baking  Value  of  Flour,"  Compt.  rend.,  10  (1911),  170-206. 

«  Minn.  Exp.  Sta.  Bull..  64  (1897). 

'  "Hat  der  gehalt  des  Weizemehles  an  Wasserloslichen  Sticksto£F  einer 
Einfluss  auf  seiner  Backwert,"  Ztschr.  Uncer.  Nahr.  Genuss.,  13  (1907), 
69-74 

*  "Les  mati^res  azote^s  solubles  comme  facteur  d'appreciation  des 
farines,"  Compt.  rend.  Acad.  Set..  166  (1913),  723-725. 


the  present  state  of  our  knowledge  regarding  the 
factors  involved  in  flour  strength,  and  is  intended  to 
serve  this  purpose  rather  than  constitute  anything 
like  a  complete  summary  of  all  the  work  which  has 
been  done  in  this  field.  Numerous  summaries  of  this 
sort  have  been  published  in  text-books  and  articles 
dealing  with  methods  of  milling  and  baking  technology, 
such  as  that  of  the  Jagos/  and  a  repetition  of  them 
here  would  serve  no  useful  purpose.  It  is  believed, 
moreover,  that  the  above  discussion  indicates  nearly 
all  of  the  view-points  from  which  the  problem  of  the 
chemistry  of  flour  strength  has  been  attacked.  The 
situation  is  very  well  expressed  by  Bailey^  when  he 
says:  "Perhaps  one  of  the  reasons  that  a  greater  de- 
gree of  success  has  not  attended  these  endeavors  is 
the  fact  that  it  has  been  attempted  to  discover  one 
constituent  (or  group  of  constituents)  which  is  the  sole 
determining  factor.  It  does  not  seem  reasonable  to 
believe  that  in  so  complex  a  substance  as  wheat  flour 
the  percentage  of  one  constituent  can  be  regarded  as 
solely  indicative  of  baking  quality.  Rather  must  we 
study  these  various  compounds  in  their  relation  to 
one  another,  in  an  effort  to  arrive  at  their  single  and 
combined  effects." 

PURPOSE     OF    THIS    INVESTIGATION 

In  a  series  of  investigations  of  the  various  factors 
which  may  influence  the  strength  of  wheat  flour,  now 
in  progress  in  the  Division  of  Agricultural  Chemistry 
of  the  University  of  Minnesota,  it  was  proposed  to 
study  the  chemical  constitution  of  the  various  proteins 
in  flour  with  a  view  toward  ascertaining  more  defi- 
nitely than  has  yet  been  done,  whether  or  not  the  pro- 
teins of  a  strong  flour  may  differ  in  their  chemical 
constitution  from  those  of  a  weak  flour,  since  the 
physical  properties  of  their  glutens  are  found  to  differ 
so  markedly. 

Wood,^  in  1907,  following  Osborne  and  Harris' 
modification  of  Hausmann's  method,  subjected  samples 
of  gliadin  and  crude  gluten  (composed  chiefly  of  gliadin 
and  glutenin)  of  flours  of  different  strength,  to  hydrolysis 
for  8  hours  with  strong  hydrochloric  acid.  He  then 
steam-distilled  the  products  of  hydrolysis  with  magnesia 
and  determined  the  percentage  of  nitrogen  given  off 
as  ammonia.  Finding  a  close  agreement  in  the  different 
samples    he    concluded   that    gliadin    and    glutenin.  of 

1  Loc.  cit. 

2  "Relation  of  the  Composition  of  Flour  to  Baking  Quality,"  Canadian 
Miller  and  Cerealisf,  5  (1913),  208-209. 

'  Loc.  cit. 


all  wheat  flours  are  of  the  same  chemical  composition, 
since  the  work  of  Wood/  a  more  detailed  method  of 
protein  analysis,  which  gives  further  insight  into  the 
constitution  of  the  protein  molecule  and  is  capable  of 
yielding  quantitative  results,  has  been  presented  by 
Van  Slyke,^  who  has  incidentally  shown  that  the 
hydrolysis  of  gliadin  with  strong  hydrochloric  acid 
is  not  complete  at  the  end  of  8  hours.  It  was  therefore 
decided  to  make  further  study  of  the  chemical  con- 
stitution of  flour  proteins  in  the  light  of  better  methods 
of  analysis  now  available. 

METHODS      OF      STUDYING      CHEMICAL      COMPOSITION      OF 
PROTEINS 

It  has  been  shown  repeatedly  that  for  practical 
considerations  all  of  the  nitrogen  of  flours  of  the  higher 
milling  grades  may  be  regarded  as  in  the  proteins. 
The  chemical  structure  of  the  proteins  has  been  clearly 
demonstrated  by  Fischer^  and  a  host  of  other  workers 
since,  so  that  it  needs  no  elaborate  discussion  here. 
Briefly  stated,  the  facts  appear  to  be  that  the  protein 
molecule  is  made  up  of  a  number  of  amino  acids,  there 
being  some  i8  or  20  of  these  which  occur  in  natural 
proteins.  These  are  probably  linked  together  by 
anhydride  combinations  between  the  amino  group  of 
one  amino  acid  and  the  carboxyl  group  of  another. 
This  is  indicated  by  the  nature  of  the  products  formed 
(amino  acids)  when  the  protein  is  subjected  to  hydroly- 
sis. Moreover,  it  appears  that  the  characteristic 
chemical  and  physical  nature  of  individual  proteins 
depends  largely  on  the  nature  and  number  of  the 
various  amino  acids  of  which  they  are  composed. 
In  a  comparison  of  the  chemical  constitution  of  proteins, 
then,  it  is  necessary  to  split  the  molecule  by  hydrolysis 
into  its  "bausteine"  (characteristic  units)  and  determine 
the  relative  proportions  of  these  which  are  formed  in 
each  case.  There  is  no  known  method  of  ascertaining 
the  exact  manner  in  which  these  units  are  grouped 
together  in  the  various  proteins,  since  even  the  sensi- 
tive anaphylaxis  reaction  is  not  specific  in  the  case  of 
many  vegetable  proteins,  as  has  been  demonstrated 
by    Wells    and    Osborne,^    who    found    that    animals 

1  Loc.  cit. 

'  "The  Analysis  of  Proteins  by  the  Determination  of  the  Chemical 
Groups  Characteristic  of  the  Dififerent  Amino  Acids,"  J.  Biol.  Chem.,  10 
(1911),    15-55. 

•  "Untersuchungen  uber  AminosSuren,  Polypeptide  und  Proteine," 
Berlin,  1899-1906 

*  "Is  the  Specificity  of  the  Anaphylaxis  Reaction  Dependent  on  the 
Chemical  Constitution  of  the  Proteins  or  on  Their  Biological  Relations? 
The  Biological  Reactions  of  the  Vegetable  Proteins.  II,"  Jour.  Infect. 
Dis..  la   (1913),  341-358. 


9 
sensitized  with  gliadin  of  either  wheat  or  rye  will  react 
with  hordein  of  barley,  a  protein  known  to  have  a 
different  chemical  constitution,  and  that  gliadin  and 
glutenin,  known  to  be  different  as  regards  the  relative 
proportions  of  the  various  amino  acids  in  their  molecule, 
react  anaphylactically  with  each  other. 

METHOD  USED  IN  DETERMINING  PRODUCTS  OF  PROTEIN 
HYDROLYSIS 

Van  Slyke's  method  gives  the  most  detailed  insight 
into  the  protein  molecule  of  any  known  method  which, 
at  the  same  time,  indicates  quantitatively  the  dis- 
tribution of  its  component  units.  Accordingly,  the 
Van  Slyke  method,  in  some  cases  slightly  modified^ 
was  used  in  this  investigation.  The  method,  which 
is  an  extension  of  the  principle  of  the  Hausmann 
method,  consists  of  a  division  of  the  protein  molecule 
into  various  groups,  or  units,  after  prolonged  hy- 
drolysis with  hydrochloric  acid,  and  the  determination 
of  the  pefcentage  of  nitrogen  in  each  individual  group, 
thus  ascertaining  the  distribution  of  the  total  nitrogen 
in  the  protein.  Briefly,  the  groups  determined  are: 
(i)  ammonia  or  amide  nitrogen,  which  is  considered 
to  be  derived  from  — CONH2  or  — CONHOC— 
groups  linked  to  the  carboxyl  groups  of  the  dicarboxylic 
acids  in  the  protein  molecule  (glutamic  and  aspartic 
acids);  (2)  humin  nitrogen,  from  the  dark-colored 
pigment  and  slight  amount  of  insoluble  matter  always 
formed  in  the  hydrolytic  products  of  acid  hydrolysis 
of  proteins;  (3)  the  amino  nitrogen  of  the  mono-amino 
acids,  which  corresponds  to  all  of  the  mono-amino 
acids  excepting  proline  and  oxy-proline;  (4)  the  non- 
amino  nitrogen  of  the  mono-amino  acids,  which  corre- 
sponds to  the  proline  and  oxy-proline;  and  (5)  to  (8) 
the  nitrogen  corresponding  to  each  of  the  individual 
di-amino  acids,  i.  e.,  arginine,  lysine,  histidine,  and 
cystine,  respectively.  Thus,  eight  units  of  the  protein 
molecule  may  be  estimated  quantitatively,  the  de- 
termination of  histidine  nitrogen  and  lysine  nitrogen 
being  subject  to  a  larger  experimental  error  than  the 
other  units,  which  may  be  determined  with  the  exact- 
ness  required   by   ordinary   quantitative   procedure. 

FLOURS    USED    IN    THE    INVESTIGATION 

Eight  flours  of  the  higher  grades  (as  separated  in  the 
process  of  milling)  from  various  sources  and  of  varying 
baking  qualities  were  selected  for  the  preliminary 
work.  Their  sources  and  relative  baking  values,  as 
measured  by  loaf  volume,  are  indicated  in  Table  I. 


gia-1 


0^  00  r^  00 1^  OS  00 


O 


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^'    M  H  e  >7  o 


00  — '  —  — I  c^  00  o\ 
-<  tN  <N  (N  CM  — -. 


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THE    PRODUCTS    OF   'PROTEIN    HYDROLYSIS    FROM    ENTIRE 
FLOUR 

Osborne^  and  his  associates  have  shown  that  there 
are  five  proteins  present  in  flour,  viz.,  gliadin,  glutenin, 
albumin,  globulin  and  proteose,  the  latter  being  of 
little  significance.  The  first  two  named  compose  the 
gluten,  already  referred  to,  while  the  others  are  soluble 
in  dilute  salt  solutions  and  are,  for  the  most  part, 
removed  in  the  familiar  process  of  "washing  out" 
the  gluten. 

Since  the  proteins  are,  for  all  practical  considerations, 
the  only  nitrogen  compounds  in  the  higher  grade 
flours,  it  was  decided  to  submit  first,  in  all  cases,  a 
sample  of  the  entire  flour  to  prolonged  hydrolysis  with 
strong  hydrochloric  acid,  and  determine  the  distribu- 
tion of  nitrogen  in  the  various  units.  Should  the  re- 
sults vary  in  different  flours,  it  would  be  necessary  to 
obtain  the  different  proteins  and  ascertain  their  com- 
position, in  a  similar  manner.  If  they  should  show  the 
same  chemical  constitution  then  they  must  be  present 
in  the  flour  in  varying  amounts  to  account  for  the  differ- 
ence when  analyzed  collectively,  as  is  done  in  the 
hydrolysis  of  the  entire  flour.  That  the  latter  is  true 
has,  of  course,  been  concluded  by  numerous  investi- 
gators who  have  extracted  flour  proteins  with  specific 
solvents  and  have  found  their  amounts  to  vary  widely 
in  different  flours.  The  solvents  most  frequently  used 
are:  (i)  alcohol — varying  from  50  to  80  per  cent,  and 
(2)  neutral  salt  solutions  of  different  concentrations. 
The  former  was  at  first  thought  to  extract  only  gliadin 
while  the  latter  was  considered  to  remove  only  albumin, 
globulin  and  proteose.  Owing  to  the  fact  that  solu- 
tions of  varying  strengths  and  different  methods  of 
extraction  have  been  employed  by  different  investi- 
gators, however,  their  results  often  disagree  widely, 
and  in  many  cases  even  fail  to  support  the  same  general 
conclusions.  Furthermore,  it  has  been  found  that  the 
solvents  mentioned  above  are  not  as  specific  as  was 
formerly  supposed,  and  that  alcohol  extracts  not  only 
gliadin  but  also  considerable  of  the  "soluble  proteins," 
the  material  so  extracted  depending  on  the  strength  of 
the  alcohol,  while  salt  solutions  extract  some  gliadin 
as  well  as  albumin  and  globulin,  according  to  the  con- 
centration of  the  solution.  Other  physico-chemical 
factors  undoubtedly  enter  as  well.     Olson^  states  that 

1  "The  Vegetable  Proteins"  (1912),  Plimmer's,  Monograph,  London, 
New  York,  etc. 

^  "Quantitative  Estimation  of  Salt-Soluble  Proteins  in  Wheat  Flour." 
J.  Ind.  Eng.  Chem.,  6  (1914),  212. 


"the  amount  of  gliadin  extracted  by  i  per  cent  sodium 
chloride  solution  approximately  amounts  to  about 
29  per  cent  of  the  total  proteids,"  and  "the  nitrogen 
bodies  soluble  in  salt  solution  are  partly  or  wholly 
soluble  in  diluted  alcohols  varying  with  the  concentra- 
tion of  sodium  chloride  used."  That  a  study  of  the 
products  of  hydrolysis  of  the  flour  proteins  both 
collectively  and  individually  can  furnish  an  indica- 
tion of  the  proportions  of  these  proteins  in  the  flour, 
providing  there  is  no  difference  in  the  chemical  con- 
stitution of  the  same  proteins  in  different  flours,  is 
evident  from  the  following  considerations:  the  per- 
centage of  ammonia  nitrogen  yielded  on  the  hydrolysis 
of  the  individual  proteins  of  wheat  flour  varies  as 
follows,  according  to  Osborne,  gliadin  24.5,  glutenin 
18.8,  leucosin  (albumin  of  flour)  6.8,  and  globulin, 
7.7.  Since  the  figures  for  the  ammonia  nitrogen 
show  wider  variation  than  do  those  of  any  other  units, 
and  since  also  the  estimation  of  this  unit  is  probably 
accompanied  by  less  error  than  that  of  any  of  the 
others,  it  may  be  supposed  that  its  estimation  in-  the 
proteins  taken  collectively  and  individually  will 
indicate  closely  the  relative  amounts  of  the  various 
proteins  present,  providing,  as  mentioned  before,  the 
same  proteins  of  different  flours  do  not  vary  in  their 
chemical  constitution. 

In  determining  the  distribution  of  nitrogen  in  the 
entire  flour,  lo-gram  samples  were  hydrolyzed  for 
48  hours,  and  the  "Hausmann"  units  determined. 
In  the  case  of  the  entire  flour  the  presence  of  a  large 
amount  of  starch  occasions  a  voluminous  precipitate 
of  "humin"  material  which  made  it  impractical  to 
attempt  a  determination  of  all  the  units  of  the  Van 
Slyke  method,  since  a  large  enough  sample  could  not 
be  used  to  insure  the  estimation  of  the  smaller  units 
with  sufficient  accuracy  that  the  figures  would  be 
of  much  significance.  The  instructions  of  Van  Slyke 
regarding  the  conditions  for  precipitating  and  washing 
the  bases,  however,  were  carefully  followed. 

In  determining  the  total  nitrogen  in  the  hydrolyzed 
mixture,  the  presence  of  large  amounts  of  "humin" 
substances  resulting  from  the  carbohydrates,  and  small 
amounts  of  fat,  necessitated  the  slight  modification  of 
Van  Slyke's  method  suggested  by  Gortner.^  The 
above    mentioned   substances    make   it   impossible   to 

1  "Studies  on  the  Chemistry  of  Embryonic  Growth.  I.  Certain 
Changes  in  the  Nitrogen  Ratios  of  Developing  Trout  Eggs,"  J.  Am.  Chem. 
Soc,  36  (1913).  632-645. 


13 
obtain  an  aliquot  until  after  they  have  been  removed 
in  the  processes  of  determining  the  ammonia  and  humin 
nitrogen.  Consequently,  the  hydrolyzed  mixture  is 
evaporated  in  vacuo  to  remove  most  of  the  hydro- 
chloric acid.  The  ammonia  is  distilled  off  as  in  the 
Van  Slyke  process  (without  removing  the  material 
from  the  distillation  flask  from  which  the  acid  was 
evaporated  off),  collected  in  standard  acid,  and  esti- 
mated by  titration;  the  humin  filtered,  washed,  and 
submitted  to  Kjeldahl  analysis  for  nitrogen,  and  total 
nitrogen  determined  in  aliquot  portions  of  the  filtrate 
from  the  humin.  This,  added  to  the  ammonia  nitrogen 
and  the  humin  nitrogen,  gives  the  total  nitrogen  in 
the  hydrolyzed  sample.  No  correction  was  made 
in  any  of  the  analyses  for  the  solubilities  of  the  bases 
in  the  solutions  from  which  they  were  precipitated, 
since  the  same  conditions  were  observed  in  all  cases 
and  the  results  are  strictly  comparable. 

The  results  given  in  Table  I  were  obtained  from  the 
analyses  of  the  eight  samples  of  flour  by  the  above- 
described  process.  The  different  flours  vary  signifi- 
cantly with  respect  to  the  ammonia  nitrogen  yielded 
on  hydrolysis.  The  basic  nitrogen  or  nitrogen  of  the 
diamino  acids  also  shows  a  slight  variation,  this  being 
inversely  as  the  variation  in  ammonia  nitrogen.  The 
variations  shown  in  the  table  are  much  greater  than 
could  possibly  be  due  to  experimental  error  and  were 
confirmed  by  repeated  determinations.  Hence,  there 
can  be  no  doubt  that  these  variations  show  actual 
characteristic  differences  in -the  nitrogen  distribution 
in  the  different  samples. 

THE  DISTRIBUTION  OF  NITROGEN  IN  GLIADIN,  GLUTENIN, 
AND  SOLUBLE  PROTEINS 

Hydrolysis  of  the  entire  flour  having  shown  character- 
istic differences  in  the  composition  of  the  entire  protein 
material  contained  in  them,  it  appeared  to  be  necessary 
to  establish  as  definitely  as  possible  whether  or  not 
the  chemical  constitution  of  -the  various  individual 
proteins  is  the  same  in  different  flours.  For  this 
purpose  two  flours  which  differed  widely  in  their 
origin,  total  nitrogen  content,  and  baking  strength  were 
selected.  Flour  B401  is  a  typical  Minnesota  patent 
flour,  milled  from  northern  spring  wheat,  of  fairly 
high  nitrogen  content  and  of  good  baking  strength, 
while  B438  is  a  patent  biscuit  flour,  made  from  a  softer 
Missouri  wheat,  low  in  total  nitrogen  and  of  poor 
baking    strength.     Gliadin    was    extracted    from    the 


14 
gluten  of  the  flours  with  alcohol  and  carefully  purified 
by  pouring  the  concentrated  syrup  from  the  clear 
alcoholic  extract  alternately  into  large  volumes  of  water 
and  strong  alcohol  and  finally  digesting  with  absolute 
alcohol  and  ether,  according  to  the  method  of  Osborne. 
Glutenin  was  also  prepared  according  to  Osborne's 
method  which  consists,  briefly,  of  dissolving  the  residue 
left  after  the  alcohol  extraction  of  the  crude  gluten  in  a 
dilute  solution  of  potassium  hydroxide,  neutralizihg 
with  hydrochloric  acid  to  precipitate  the  glutenin, 
decanting  the  liquid  and  further  extracting  the  pre- 
cipitate repeatedly  with  alcohol  to  remove  the  re- 
maining gliadin;  finally  digesting  with  absolute  alco- 
hol and  ether.  The  preparations  of  glutenin  in  this 
work  were  not  pure,  being  contaminated  by  small 
quantities  of  carbohydrates,  owing  to  lack  of  facilities 
for  obtaining  clear  extracts  and  filtrates  at  the  time, 
but  it  is  believed  that  all  other  nitrogen-containing 
bodies  were  removed,  and  that  the  preparations  served 
the  purpose  of  the  investigation,  namely,  to  ascertain 
whether  there  was  any  appreciable  difference  in  the 
chemical  constitution  of  the  pure  proteins.  Consider- 
able quantities  of  each  of  the  two  flours  were  then 
extracted  with  i  per  cent  salt  solution,  the  extracts 
were  filtered  as  clear  as  possible  and  concentrated 
in  vacuo.  These  extracts  and  weighed  quantities  of 
the  gliadin  and  glutenin  were  then  hydrolyzed  for  48 
hours  with  strong  HCl.  The  gliadin  and  glutenin 
were  analyzed  according  to  the  Van  Slyke  method, 
while  only  ammonia  nitrogen  was  determined  in  the 
case  of  the  soluble  proteins.  From  the  results  shown 
in  Table  II  (i,  2,  and  3)  it  is  readily  seen  that,  after 
making  allowance  for  the  limits  of  experimental  error 
of  the  method,  there  is  no  apparent  difference  in  the 
chemical  constitution  of  the  proteins  of  typical  strong 
and  weak  flours  of  the  same  market  grade. 

THE     DISTRIBUTION     OF     NITROGEN     IN     CRUDE     GLUTEN 

More  complete  evidence  that  the  gluten-forming 
proteins  are  of  the  same  chemical  constitution  in  differ- 
ent flours  was  obtained  by  analyzing  thoroughly 
washed  crude  glutens  of  three  flours  of  widely  differing 
characteristics.  The  same  two  flours  as  in  the  immedi- 
ately preceding  experiments  were  used,  and  in  addition, 
B444,  a  Kansas  flour  of  exceedingly  high  nitrogen  and 
gluten  content,  but  of  low  baking  strength,  as  shown 
in  Table  I.  The  results,  obtained  from  the  complete 
Van>Slyke  process  as  applied  to  the  crude  glutens  from 
these  three  flours,  appear  in  Table  II  (4)  and  indicate 


15 
that  not  only  are  the  gluten-forming  proteins  in  flours 
of  widely  differing  baking  qualities  of  the  same  chemical 
constitution,  but  the  ratio  of  gliadin  to  glutenin  is 
probably  the  same,  or  very  nearly  so,  in  flours  of  the 
same  market  grade  but  very  different  baking  strengths. 
With  respect  to  this  latter  point,  it  may  be  said  that 
since,  as  is  shown  above,  gliadin  yields  26  per  ceut  of 
its  nitrogen  as  ammonia  nitrogen  after  hydrolysis, 
while  glutenin  yields  only  16  per  cent  of  its  nitrogen 
in  this  fraction,  the  determination  of  ammonia  nitrogen 
of  the  hydrolyzed  glutens  will  certainly  indicate  any 
significant  variation  in  the  ratio  of  gliadin  to  glutenin 
in  different  flours,  although  the  limits  of  experimental 
error  are  not  narrow  enough  to  indicate  very  small 
variations  in  this  ratio.  .  The  data  in  Table  II  (4) 
indicate  very  clearly,  therefore,  that  there  is  no  signifi- 
cant variation  in  the  gliadin-glutenin  ratio  in  flours 
of  such  widely  varying  baking  strength  as  those  used 
in  this  investigation. 

THE      SIGNIFICANCE      OF      THE      SOLUBLE      PROTEINS      IN 
AFFECTING   THE   NITROGEN   DISTRIBUTION  IN   FLOUR 

It  is  evident  that  the  differences  in  the  percentages 
of  ammonia  nitrogen  and  basic  nitrogen  yielded  on  the 
hydrolysis  of  the  several  entire  flours,  as  shown  in 
Table  I,  cannot  be  accounted  for  as  being  due  to 
differences  in  chemical  composition  of  the  individual 
•  proteins  since  it  has  been  clearly  shown  that  these 
have  the  same  chemical  constitution. 

It  was  thought  at  first  that  the  varying  percentages 
of  starch  in  the  different  flours  might  cause  differences 
in  the  percentage  of  ammonia  nitrogen,  since  Mann,^ 
in  1906,  states  "if  in  addition  to  the  carbohydrate, 
ammonia  or  other  nitrogenous  substances  are  in  solu- 
tion, theii  the  humins  combine  with  the  ammonia  and 
thereby  become  nitrogenous."  In  order  to  ascertain 
whether  varying  proportions  of  starch  would  influence 
the  results  obtained  by  the  Van  Slyke  method  as  used 
in  these  investigations,  a  sample  of  the  flour  B401, 
to  which  had  previously  been  added  20  per  cent  of 
its  weight  of  wheat  starch,  was  hydrolyzed  and  the 
distribution  of  nitrogen  in  the  products  of  hydrolysis 
determined.  There  was  no  significant  change  in  the 
percentage  of  ammonia  nitrogen  when  compared  with 
the  sample  to  which  no  starch  was  added,  although 
there  was  a  very  noticeable  increase  in  humin  N  and 
a  corresponding  decrease  in  basic  N,  as  shown  in  Table 
III. 

>  "Chemistry  of  the  Proteids,"  New  York.  1906. 


BIOGRAPHICAL 

Morris  J.  Blish  was  born  April  21,  1889,  at  Lincoln, 
Nebraska.  He  attended  the  public  schools  there, 
through  the  seventh  grade.  He  then  moved  to  Omaha, 
Nebraska,  where  he  graduated  from  the  Omaha  High 
School  in  1906.  After  working  in  one  of  the  Omaha 
banks  for  a  year,  he  entered  the  University  of  Nebraska 
in  the  fall  of  1907,  specializing  in  chemistry  and  grad- 
uated with  the  B.Sc.  degree  in  February,  1912.  Enroll- 
ing in  the  graduate  school  of  the  University  of  Nebraska, 
he  received  the  A.M.  degree  in  agricultural  chemistry, 
June,  1913,  thesis  work  having  been  on  soil  chemistry 
under  the  direction  of  Dr.  F.  J.  Alway.  He  received 
an  appointment  as  research  assistant  in  agricultural 
chemistry  at  the  University  of  Minnesota,  and  enrolled 
in  the  graduate  college  of  that  university  in  Septem- 
ber, 1 9 13,  working  under  the  direction  of  Prof.  R.  W. 
Thatcher.  He  received  the  Ph.D.  degree  in  June, 
191 5,  thesis  work  having  been  on  the  chemical  consti- 
tution of  the  proteins  of  wheat  flour,  in  relation  to 
"baking  strength." 


PUBLICATIONS 

1.  On  the  Distribution  and  Composition  of  the 
Humus  of  the  Loess  Soils  of  the  Transition  Region. 

2.  On  the  Origin  of  the  Humin  Formed  by  the 
Acid  Hydrolysis  of  Proteins.  (In  collaboration  with 
Dr.  R.  A.  Gortner.) 

3.  Concerning  the  Identity  of  the  Proteins  Ex- 
tracted from  Wheat  Flour  by  the  Usual  Solvents.  (In 
collaboration  with  C.  H.  Bailey.) 


